VALIDATION DATA. Multiplex CVD II 96 96

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1 VALIDATION DATA Multiplex CVD II 96 96

2 Table of contents 1. INTRODUCTION Technology Quality Controls Data analysis 3 2. PERFORMANCE CHARACTERISTICS Sample types Analytical Measurement 4 Detection limit 4 High dose hook effect 4 Measuring range Precision 8 Repeatability 8 Reproducibility Analytical Specificity 8 Assay readout specificity 8 Endogenous interference Scalability 9 3. REFERENCES 10 TECHNICAL SUPPORT For technical support, please contact us at support@olink.com or

1. Introduction Proseek Multiplex CVD II 96 96 is a reagent kit measuring 92 cardiovascular disease (CVD) related human protein biomarkers simultaneously.

1 TECHNOLOGY The Proseek Multiplex reagents are based on the Proximity Extension Assay (PEA) technology 1-2, where 92 oligonucleotide labeled antibody probe pairs are allowed to bind to their

A PCR reporter sequence is formed by a proximity dependent DNA polymerization event, amplified, and subsequently detected and quantified using real-time PCR.

1.2 QUALITY CONTROLS Internal and external controls have been developed by Olink for data normalization and quality control purposes.

protocol (see Figure 1). The internal controls are added to each sample and include two Immunoassay controls, one Extension control and one Detection control.

3 1. Introduction Proseek Multiplex CVD II is a reagent kit measuring 92 cardiovascular disease (CVD) related human protein biomarkers simultaneously. The analytical performance of the product has been carefully validated and the results are presented below. 1.1 TECHNOLOGY The Proseek Multiplex reagents are based on the Proximity Extension Assay (PEA) technology 1-2, where 92 oligonucleotide labeled antibody probe pairs are allowed to bind to their respective target protein present in the sample. A PCR reporter sequence is formed by a proximity dependent DNA polymerization event, amplified, and subsequently detected and quantified using real-time PCR. The assay is performed in a homogeneous 96 well format without any need for washing steps, see Figure QUALITY CONTROLS Internal and external controls have been developed by Olink for data normalization and quality control purposes. These controls have been designed to enable monitoring of the technical assay performance, as well as the quality of individual samples, providing information at each step of the Proseek Multiplex protocol (see Figure 1). The internal controls are added to each sample and include two Immunoassay controls, one Extension control and one Detection control. The Immunoassay controls (two non-human proteins) monitor all three steps starting with the immunoreaction. The Extension Control (an antibody linked to two matched oligonucleotides for immediate proximity independent of antigen binding) monitors the extension and readout steps and is used for data normalization across samples. Finally, the Detection control (a synthetic double-stranded template) monitors the readout step. Samples for which one or more of the internal control values deviate from a pre-determined range will be flagged and may be removed before statistical analysis. An external control, inter-plate control (IPC), is included on each plate and used in a second normalization step. This control is made up of a pool of probes similar to the Extension control (Ext Ctrl), but generated with 92 matching oligonucleotide pairs. Furthermore, the improves inter-assay precision and allows for optimal comparison of data derived from multiple runs. The term Normalized Protein expression (NPX) refers to normalized data as described above. 1.3 DATA ANALYSIS Data analysis was performed by employing a preprocessing normalization procedure. For each sample and data point, the corresponding Cq-value for the Extension control was substracted, thus normalizing for technical variation within one run. Normalization between runs is then performed for each assay by substracting the corresponding dcq-value for the Interplate Control (IPC) from the dcq-values generated. In the final step of the pre-processing procedure the values are set relative to a correction factor determined by Olink. The generated Normalized Protein expression (NPX) unit is on a log2 scale where a larger number represents a higher protein level in the sample, typically with the background level at around zero. Linearization of data is performed by the mathematical operation 2^NPX. Coefficient of variation (CV) calculations were performed on linearized values. IMMUNOASSAY Allow the 92 antibody probe pairs to bind to their respective proteins in your samples. EXTENSION Extend and pre-amplify 92 unique DNA reporter sequences by proximity extension. DETECTION Quantify each biomarker s DNA reporter using high throughput real-time qpcr. Immunoassay control Extension control Detection control Fig 1. Proseek Multiplex assay procedure (above) and controls (below). The internal controls enables monitoring of the three core steps in the Proseek Multiplex assay and used for quality control and data normalization. Read out is performed by using the Fluidigm Biomark or the Fluidigm Biomark HD system. Proseek Multiplex CVD II Validation Data 3

4 2. Performance characteristics 2.1 SAMPLE TYPES The ability to use different sample types was evaluated with Proseek Multiplex CVD II by collecting matched serum, EDTA, acid citrate dextrose (ACD), and sodium heparin plasma samples from 4 healthy individuals. Table 1 summarizes response values for 20 normal EDTA plasma samples expressed in NPX, as well as relative differences compared to EDTA plasma. Variations observed between responses in heparin, citrate plasma and serum, as compared to EDTA plasma, were generally small, and all assays will therefore function without limitation in these sample types. 2.2 ANALYTICAL MEASUREMENT DETECTION LIMIT Calibrator curves were determined for 91 out of 92 biomarkers simultaneously in a multiplex format. One protein biomarker (NEMO) lacked accessible recombinant antigen. Limit of detection (LOD) was defined as 3 standard deviations above background and reported in pg/ml for all assays where recombinant protein antigen was available, see Table 1 and Figure 2. A) B) NPX NPX LOD: pg/ml LLOQ: pg/ml ULOQ: pg/ml Hook: pg/ml Range: 2.11 log10 ADM LLOQ LOD: 0.48 pg/ml LLOQ: 0.48 pg/ml ULOQ: pg/ml Hook: pg/ml Range: 5.12 log10 Concentration (pg/ml) IL-18 ULOQ ULOQ LOD HIGH DOSE HOOK EFFECT The high dose hook effect is a state of antigen excess relative to the reagent antibodies, resulting in falsely lower values. In such cases, a significantly lower value can be reported which leads to misinterpretation of results. Therefore, the hook effect was determined for each analyte, here reported in pg/ml for 91 out of 92 assays, see Table 1. C) 4 2 LLOQ LOD Concentration (pg/ml) 18 MMP-7 MEASURING RANGE The analytical measuring range was defined by the lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) and reported in order of log10, see Table 1. The upper and lower limits of quantification, ULOQ and LLOQ, respectively were calculated with the following trueness and precision criteria; relative error 30% and CV 30%, of backcalculated values, and reported in pg/ml, see Table 1. Three assays with their analytical data are shown in Figure 2 and the distribution of measuring ranges of 90 assays and endogenous plasma levels are shown in Figure 3. Separate calibrator curves established for each assay may be viewed at NPX LOD: 7.63 pg/ml LLOQ: 7.63 pg/ml ULOQ: pg/ml Hook: pg/m Range: 3.61 log10 LLOQ ULOQ LOD Concentration (pg/ml) Fig 2. Calibrator curves from 3 assays and their corresponding analytical measurement data. 4 Proseek Multiplex CVD II Validation Data

5 GT ITGB1BP2 DECR1 LEP ADM AGRP PD-L2 CTSL1 STK4 BNP PIgR MERTK SLAMF7 ANG-1 CEACAM8 HAOX1 TNFRSF13B ADAM-TS13 THBS2 SOD2 SORT1 FGF-21 FGF-23 XCL1 PARP-1 HSP 27 IL27-A IL1RL2 DCN PAR-1 THPO PAPPA CD84 SPON2 VEGF-D Protein BOC TGM2 PDGF subunit B MARCO VSIG2 GDF-2 IL16 IgG Fc receptor II-b RAGE MMP-12 CD4 Dkk-1 Gal-9 ACE2 PRELP SRC SERPINA12 CXCL1 TIE2 hoscar TM IL-4RA HO-1 REN MMP-7 PSGL-1 IL-1ra CTRC TNFRSF10A IL-17D BMP-6 FS PRSS27 SCF PTX3 KIM-1 GH CCL17 IDUA LPL GLO1 LOX-1 TNFRSF11A CD40-L CA5A IL-18 PlGF GIF HB-EGF CCL3 TF PRSS8 IL-6 TRAIL-R2 FABP Protein concentration (pg/ml) Fig 3. Distribution of analytical measuring range, defined by the lower and upper limits of quantification (LLOQ-ULOQ), and normal plasma levels for 90 out of 92 analytes. Proseek Multiplex CVD II Validation Data 5

6 Table 1. Sample Types; Normalized Protein expression (NPX), Endogenous Interference, Analytical Measurement; Limit of Detection (LOD), Lower Limit of Quantification (LLOQ), Upper Limit of Quantification (ULOQ), High Dose Effect (Hook), Range and Precision indicative of assay performance are shown for 92 analytes. Not available, NA Sample types Endogenous Interference Analytical measurement Precision Normal plasma levels (NPX) Relative to EDTA plasma (%) (mg/ml) pg/ml log10 % CV Target UniProt No 10th %tile Median 90th %tile ACD Heparin Serum Haemolysate LOD LLOQ ULOQ Hook Range Intra Inter ADM (ADM) P ,4-dienoyl-CoA reductase, mitochondrial (DECR1) A disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAM-TS13) Q Q76LX Agouti-related protein (AGRP) O Alpha-L-iduronidase (IDUA) P Angiopoietin-1 (ANG-1) Q Angiopoietin-1 receptor (TIE2) Q Angiotensin-converting enzyme 2 (ACE2) Q9BYF Bone morphogenetic protein 6 (BMP-6) P Brother of CDO (Protein BOC) Q9BWV Carbonic anhydrase 5A, mitochondrial (CA5A) Carcinoembryonic antigenrelated cell adhesion molecule 8 (CEACAM8) P P Cathepsin L1 (CTSL1) P C-C motif chemokine 17 (CCL17) Q C-C motif chemokine 3 (CCL3) P CD40 ligand (CD40-L) P Chymotrypsin C (CTRC) Q C-X-C motif chemokine 1 (CXCL1) P Decorin (DCN) P Dickkopf-related protein 1 (Dkk-1) O Fatty acid-binding protein, intestinal (FABP2) P Fibroblast growth factor 21 (FGF-21) Q9NSA Fibroblast growth factor 23 (FGF-23) Q9GZV Follistatin (FS) P Galectin-9 (Gal-9) O Gastric intrinsic factor (GIF) P Gastrotropin (GT) P Growth hormone (GH) P Growth/differentiation factor 2 (GDF-2) Q9UK Heat shock 27 kda protein (HSP 27) P Heme oxygenase 1 (HO-1) P Hydroxyacid oxidase 1 (HAOX1) Q9UJM Interleukin-1 receptor antagonist protein (IL-1ra) P Interleukin-1 receptor-like 2 (IL1RL2) Q9HB Interleukin-17D (IL-17D) Q8TAD Interleukin-18 (IL-18) Q Interleukin-27 (IL27) Interleukin-4 receptor subunit alpha (IL-4RA) Q8NEV9 Q P Interleukin-6 (IL-6) P Lactoylglutathione lyase (GLO1) Q Lectin-like oxidized LDL receptor 1 (LOX-1) P Leptin (LEP) P Lipoprotein lipase (LPL) P Low affinity immunoglobulin gamma Fc region receptor II-b (IgG Fc receptor II-b) P Lymphotactin (XCL1) P Macrophage receptor MARCO (MARCO) Q9UEW Matrix metalloproteinase-12 (MMP-12) P Matrix metalloproteinase-7 (MMP-7) P Proseek Multiplex CVD II Validation Data

7 Sample types Endogenous Interference Analytical measurement Precision Normal plasma levels (NPX) Relative to EDTA plasma (%) (mg/ml) pg/ml log10 % CV Target UniProt No 10th %tile Median 90th %tile ACD Heparin Serum Haemolysate LOD LLOQ ULOQ Hook Range Intra Inter Melusin (ITGB1BP2) Q9UKP Natriuretic peptides B (BNP) P16860 NA NA NA NA NA NA NA NA NA NF-kappa-B essential modulator (NEMO) Q9Y6K NA NA NA NA NA Osteoclast-associated immunoglobulinlike receptor (hoscar) Q8IYS Pappalysin-1 (PAPPA) Q NA Pentraxin-related protein PTX3 (PTX3) P Placenta growth factor (PlGF) P Platelet-derived growth factor subunit B (PDGF subunit B) P Poly [ADP-ribose] polymerase 1 (PARP-1) P NA NA Polymeric immunoglobulin receptor (PIgR) Programmed cell death 1 ligand 2 (PD-L2) Proheparin-binding EGF-like growth factor (HB-EGF) P Q9BQ Q Pro-interleukin-16 (IL16) Q Prolargin (PRELP) P Prostasin (PRSS8) Q Protein AMBP (AMBP) P Proteinase-activated receptor 1 (PAR-1) P Protein-glutamine gammaglutamyltransferase 2 (TGM2) Proto-oncogene tyrosine-protein kinase Src (SRC) P P P-selectin glycoprotein ligand 1 (PSGL-1) Q Receptor for advanced glycosylation end products (RAGE) Q Renin (REN) P Serine protease 27 (PRSS27) Q9BQR Serine/threonine-protein kinase 4 (STK4) Q Serpin A12 (SERPINA12) Q8IW SLAM family member 5 (CD84) Q9UIB SLAM family member 7 (SLAMF7) Q9NQ Sortilin (SORT1) Q Spondin-2 (SPON2) Q9BUD Stem cell factor (SCF) P Superoxide dismutase [Mn], mitochondrial (SOD2) P Kidney injury molecule 1 (KIM-1) Q96D T-cell surface glycoprotein CD4 (CD4) P Thrombomodulin (TM) P Thrombopoietin (THPO) P Thrombospondin-2 (THBS2) P Tissue factor (TF) P TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) Tumor necrosis factor receptor superfamily member 10A (TNFRSF10A) Tumor necrosis factor receptor superfamily member 11A (TNFRSF11A) Tumor necrosis factor receptor superfamily member 13B (TNFRSF13B) O O Q9Y6Q O Tyrosine-protein kinase Mer (MERTK) Q Vascular endothelial growth factor D (VEGF-D) V-set and immunoglobulin domaincontaining protein 2 (VSIG2) O Q96IQ Proseek Multiplex CVD II Validation Data 7

2.3 PRECISION REPEATABILITY Intra-assay variation (within-run) was calculated as the mean %CV for 6 individual samples run in triplicates within each of 10 separate runs during the validation studies.

Variation calculations were performed on linearized values for 91 out of 92 analytes for which response levels could be measured in serum and normal plasma, see Table 1.

The distribution of both intraassay and inter-assay variations are shown in Figure 4. % of protein biomarkers 50 40 30 20 10 0 Intra (mean %CV 9.1) Inter (mean %CV 11.

Distribution of intra-assay and inter-assay variations of Proseek Multiplex CVD II 96 96 REPRODUCIBILITY Inter-site variations (between-site) were investigated during the validation of previous

8 2.3 PRECISION REPEATABILITY Intra-assay variation (within-run) was calculated as the mean %CV for 6 individual samples run in triplicates within each of 10 separate runs during the validation studies. Inter-assay variation (between runs) was calculated between experiments with the same operator. The reported inter-assay %CV is the average of three operators %CV. Variation calculations were performed on linearized values for 91 out of 92 analytes for which response levels could be measured in serum and normal plasma, see Table 1. Across all 91 assays, the mean intra-assay and inter-assay variations were observed to be 9.1% and 11.7%, respectively. The distribution of both intraassay and inter-assay variations are shown in Figure 4. % of protein biomarkers Intra (mean %CV 9.1) Inter (mean %CV 11.7) >40 % CV Fig 4. Distribution of intra-assay and inter-assay variations of Proseek Multiplex CVD II REPRODUCIBILITY Inter-site variations (between-site) were investigated during the validation of previous panels in betasite studies to estimate the expected variations in values between different laboratories, with different operators and using different equipment. The betasite studies have previously shown reproducibility and repeatability in line with Olink Bioscience results, and were therefore not performed for Proseek Multiplex CVD II For information on performed beta-site studies, download our Data Validation documents at ANALYTICAL SPECIFICITY ASSAY SPECIFICITY To test that the antibodies selected for use in our Proseek Multiplex CVD II assays are specific for their desired targets, we measured each assay response to all of the 92 panel-specific proteins, as well as against an additional 107 proteins (not shown). In principle, the specificity is tested by creating a test sample, consisting of a pool of antigens, which is then incubated with all 92 antibody probe pairs from the panel. Only if there is a correct match will a reporter sequence be created and serve as a template for subsequent real-time qpcr. Ten sub-pools of antigen are evaluated to cover the 92 assays in Proseek Multiplex, see Figure 5. None of the Proseek Multiplex CVD II 96x96 showed significant signal from the proteins tested NPX plex Pool 1 Pool 2 Pool 3 Pool 4 Pool 5 20 Pool 6 Pool 7 10 Pool 8 1 Pool 9 Fig 5. Assay readout specificity of the Proseek Multiplex platform. For each assay, specificity is confirmed by testing antigen sub-pools against the complete 92-plex pool as to each sub-mix. 8 Proseek Multiplex CVD II Validation Data

ENDOGENOUS INTERFERENCE Endogenous interference from heterophilic antibodies, e.g. human anti-mouse antibody (HAMA), and rheumatoid factor are known to cause problems in some immunoassays.

9 ENDOGENOUS INTERFERENCE Endogenous interference from heterophilic antibodies, e.g. human anti-mouse antibody (HAMA), and rheumatoid factor are known to cause problems in some immunoassays. Evaluation of the potential impact of this specific interference has been performed previously using a special mismatch system. The only way to generate a signal in this system is by antibody probe pairs being brought into proximity, by cross-binding substances other than antigens, e.g. heterophilic antibodies and similarly acting rheumatoid factor. No interference due to HAMA or RF could be detected for any of the samples in any of the previously tested panels, indicating sufficient blocking of these agents (data not shown). 2.5 SCALABILITY Assay performance was further evaluated with regard to scalability, meaning the capability of the Proseek Multiplex technology to maintain the same quality of performance irrespective of multiplex level. Previously, we have shown that a step-wise increase of multiplex grade (8, 24, 48, 72 and 96) does not compromise assay performance (data not shown). To further strengthen that Proseek provides consistent results, single assays for Growth Hormone (GH) and Matrix Metalloproteinase (MMP-7) were compared when run in the full Proseek Multiplex CVD II panel. The results for each assay and their observed dcq-values were plotted against the entire 96-plex reaction. The square of the correlation coefficient (R 2 ) value was generated by linear regression. MMP-7 GH 15 g/l 7.5 g/l 3.75 g/l 1.88 g/l 0.94 g/l 0.47 g/l 0.23 g/l EDTA plasma 3 2 R² = Fig 6. Endogenous interference. Levels tested for hemolysate were g/l hemoglobin. The highest hemolysate concentration translates to about 10% hemolysis. The potential impact of bilirubin, lipids and hemolysate, known interfering plasma and serum components, were evaluted at different added concentrations. An example of hemolysate levels tested is shown in Figure 6. These additions represent different patient health conditions and/or sample collection irregularities. For all assays, bilirubin and lipids could be added to concentrations corresponding to at least 8 or 10 times normal values 3, 4, respectively, without disturbing assay performance (data not shown). In 22 out of 92 assays, altered signal was observed by the addition of hemolysate. The reason is most likely due to actual analyte leaking out of the disrupted blood cells. A concentration of 15 g/l of hemolysate represents 10% hemolysis of a sample. Table 1 reports the highest concentration of hemolysate that does not have an impact on assay performance. Singleplex R² = Multiplex Fig 7. Scalability of the Proseek Mutliplex technology platform. This experiment was performed using the Proseek Multiplex CVD II panel. Human plasma samples were analyzed for two representative assays in singleplex and with the complete Proseek Multiplex CVD II panel. The observed dcq (log2) values were plotted, and the correlation coefficient R 2 value was generated by linear regression. Proseek Multiplex CVD II Validation Data 9

10 3. References 1. Assarsson E, Lundberg M, Holmquist G, Björkesten J, Bucht Thorsen S, Ekman D, Eriksson A, Rennel Dickens E, Ohlsson S, Edfeldt G, Andersson AC, Lindstedt P, Stenvang J, Gullberg M, Fredriksson S. Homogenous 96-Plex PEA Immunoassay Exhibiting High Sensitivity, Specificity, and Excellent Scalability. PLoS One April (2014). doi: /journal.pone Lundberg M, Eriksson A, Tran B, Assarsson E, Fredriksson S. Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low abundant proteins in human blood. Nucleic Acid Res June (2011). doi: /nar/gkr Proseek Multiplex CVD II Validation Data

12 For Research Use Only. Not for Use in Diagnostic Procedures. This product includes a license for non-commercial use of Proseek products. Commercial users may require additional licenses. Please contact Olink Proteomics AB for details. There are no warranties, expressed or implied, which extend beyond this description. Olink Proteomics AB is not liable for property damage, personal injury, or economic loss caused by this product. The following trademarks are owned by Olink AB: Olink, Olink Bioscience, Proseek. This product is covered by several patents and patent applications including US 6,511,809, US 7,306,904 and related US and foreign patents. This product is sold under license from PHRI Properties, Inc. and may be used under PHRI Properties patent rights outside the field of human in vitro diagnostics. Components in the Proseek Multiplex Probe Kit utilise Lightning-Link technology and are provided under license from Innova Biosciences. Copyright 2016 Olink Proteomics AB. All third party trademarks are the property of their respective owners. 1016, v1.3, Olink Proteomics Dag Hammarskjölds v. 52B SE Uppsala, Sweden

DATA PACKAGE. Multiplex CVD I 96 96

DATA PACKAGE. Multiplex CVD I 96 96 DATA PACKAGE Multiplex CVD I 96 96 Table of contents 1. INTRODUCTION 3 1.1 Technology 3 1.2 Data analysis 3 2. PERFORMANCE CHARACTERISTICS 4 2.1 Sample types 4 2.2 Analytical Measurement 4 Detection limit